The overall objectives of this project are to investigate further two actions of the barbiturates on the cholinergic system, inhibition of high affinity choline uptake and inhibition of acetylcholine (ACh) release, which have been demonstrated after in vivo administration of these drugs. We plan to study each of these actions in detail in rats. First, to determine if they are related to the sedative-hypnotic effect, we will test the stereospecificity of the inhibition of choline uptake and ACh release with isomers of pentobarbital and secobarbital and we will see if tolerance develops to these effects after chronic barbiturate administration to the animals. The inhibition of choline uptake is measured in hippocampal synaptosomes in vitro after in vivo drug administration. We plan to determine if this in vivo effect occurs at the nerve ending, at the cholinergic cell body (in the medial septum) or indirectly by an action elsewhere in the brain. We also intend to explore possible protocols for eliciting the inhibition of choline uptake by barbiturates added in vitro which should be possible if the drugs do act at the level of the nerve ending. Inhibition of ACh release will be studied in a new in vitro model in which a transverse slice of the hippocampus is stimulated via electrical impulses to the attached fimbria. It is thought that this will be a better model of the in vivo situation than K-stimulation of slices and will be more readily manipulated than an in vivo method. In addition to separate studies on these two functions we wish to explore the relationships of choline uptake to ACh release and to determine if the inhibition of choline uptake by barbiturates can result in an inhibition of ACh release or if there is a separate effect on the transmitter release process.